Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, Texas, USA.
Pharmacogenet Genomics. 2012 Jul;22(7):525-37. doi: 10.1097/FPC.0b013e32835366f6.
Aspirin (ASA), a major antiplatelet and cancer-preventing drug, irreversibly blocks the cyclooxygenase (COX) activity of prostaglandin H synthase-1 (PGHS-1). Considerable differences in ASA effectiveness are observed between individuals, and some of this variability may be due to PGHS-1 protein variants. Our overall aim is to determine which, if any, of the known variants in the mature PGHS-1 protein lead to functional alterations in COX catalysis or inhibition by ASA. The present study targeted four PGHS-1 variants: R53H, R108Q, L237M, and V481I.
Wild-type human PGHS-1 and the four polymorphic variants were expressed as histidine-tagged, homodimeric proteins in insect cells using baculovirus vectors, solubilized with a detergent, and purified by affinity chromatography. The purified proteins were characterized in vitro to evaluate COX and peroxidase (POX) catalytic parameters and the kinetics of COX inhibition by ASA and NS-398.
Compared with the wild type, several variants showed a higher COX/POX ratio (up to 1.5-fold, for R108Q), an elevated arachidonate Km (up to 1.9-fold, for R108Q), and/or a lower ASA reactivity (up to 60% less, for R108Q). The decreased ASA reactivity in R108Q reflected both a 70% increase in the Ki for ASA and a 30% decrease in the rate constant for acetyl group transfer to the protein. Computational modeling of the brief ASA pulses experienced by PGHS-1 in circulating platelets during daily ASA dosing predicted that the 60% lower ASA reactivity in R108Q yields a 15-fold increase in surviving COX activity; smaller, approximately two-fold increases in surviving COX activity were predicted for L237M and V481I. NS-398 competitively inhibited COX catalysis of the wild type (Ki=6 µmol/l) and inhibited COX inactivation by 1.0 mmol/l ASA in both the wild type (IC50=0.8 µmol/l) and R108Q (IC50=2.1 µmol/l).
Of the four PGHS-1 variants examined, R108Q exerts the largest functional effects, with evidence for impaired interactions with a COX substrate and inhibitors. As Arg108 is located on the protein surface and not in the active site, the effects of R108Q suggest a novel, unsuspected mechanism for the modulation of the PGHS-1 active site structure. The lower intrinsic ASA reactivity of R108Q, V481I, and L237M, combined with the rapid hydrolysis of ASA in the blood, suggests that these variants decrease the antiplatelet effectiveness of the drug. These PGHS-1 variants are uncommon but ASA is used widely; hence, a considerable number of individuals could be affected. Further examination of these and other PGHS-1 variants will be needed to determine whether PGHS-1 genotyping can be used to personalize anti-COX therapy.
阿司匹林（ASA）是一种主要的抗血小板和防癌药物，可不可逆地阻断前列腺素 H 合酶-1（PGHS-1）的环氧化酶（COX）活性。个体之间ASA 的有效性存在显著差异，其中一些差异可能归因于 PGHS-1 蛋白变体。我们的总体目标是确定成熟 PGHS-1 蛋白中已知的变体中哪些（如果有）导致 COX 催化或 ASA 抑制的功能改变。本研究针对四个 PGHS-1 变体：R53H、R108Q、L237M 和 V481I。
使用杆状病毒载体在昆虫细胞中表达野生型人 PGHS-1 和四个多态变体作为组氨酸标记的同源二聚体蛋白，用去污剂溶解，并通过亲和层析纯化。体外纯化的蛋白用于评估 COX 和过氧化物酶（POX）催化参数以及 ASA 和 NS-398 对 COX 抑制的动力学。
与野生型相比，几种变体显示出更高的 COX/POX 比值（高达 1.5 倍，对于 R108Q）、升高的花生四烯酸 Km（高达 1.9 倍，对于 R108Q）和/或更低的 ASA 反应性（高达 60%，对于 R108Q）。R108Q 中较低的 ASA 反应性反映了 ASA 的 Ki 增加了 70%，以及乙酰基转移到蛋白的速率常数降低了 30%。在循环血小板中 PGHS-1 每天接受 ASA 给药期间经历的短暂 ASA 脉冲的计算模型预测，R108Q 中较低的 60% ASA 反应性导致存活的 COX 活性增加 15 倍；对于 L237M 和 V481I，预测存活的 COX 活性增加约两倍。NS-398 竞争性抑制野生型 COX 催化（Ki=6 μmol/l），并抑制野生型（IC50=0.8 μmol/l）和 R108Q（IC50=2.1 μmol/l）中 1.0 mmol/l ASA 对 COX 失活的抑制作用。
在所检查的四个 PGHS-1 变体中，R108Q 产生最大的功能影响，证据表明与 COX 底物和抑制剂的相互作用受损。由于 Arg108 位于蛋白质表面而不是活性部位，因此 R108Q 的作用表明了一种调节 PGHS-1 活性部位结构的新的、意想不到的机制。R108Q、V481I 和 L237M 较低的内在 ASA 反应性，加上血液中 ASA 的快速水解，表明这些变体降低了药物的抗血小板作用。这些 PGHS-1 变体并不常见，但 ASA 广泛使用；因此，相当多的人可能会受到影响。需要进一步研究这些和其他 PGHS-1 变体，以确定 PGHS-1 基因分型是否可用于个性化 COX 治疗。
